Effects Of Alkaline Salt On Seedling Growth Of Brassica Napus L. And Analysis Of Salt Tolerance Candidate Genes

China is one of the major rapeseed producing countries in the world.In recent years,the planting area in China has decreased significantly,exacerbating the gap in China’s edible oil.However,there is a large planting area in Huanghuaihai of China for rapeseed,which has a wide area and more idle saline land.Under the severe pressure that oil production cannot meet the consumer demand,how to make full use of saline land to expand rapeseed planting area and increase rapeseed yield is an important issue that needs to be solved urgently in rapeseed production.However,there is currently little research on the salt tolerance mechanism of rapeseed.Therefore,it is urgent to isolate the salt tolerance genes of rapeseed and apply them to salt tolerance breeding,which is of great significance to increase the rapeseed planting area and improve its productivity.In this study,the B.napus line 2205（salt-tolerant）and 1423（sensitive salt）were used to study the changes of cell morphology and physiological indexes of seedlings before and after treatment by artificial simulation of alkaline salt stresses.Transcript sequencing technology was adopted to analyze the gene expression,and identify the alkaline salt tolerance related genes in B.napus.Combined with the information of QTLs related to salt-tolerance in the previous study,preliminary confirm the genes related to alkaline salt tolerance,clone and analyze expression of candidate genes,this study will lay a good foundation for the research on molecular breeding and mechanism of salt-tolerance in B.napus.The main results of this study are as follows:1.Effects of alkaline salt stress on leaf cell structure of B.napusThe salt tolerant line 2205 and the salt sensitive line 1423 of B.napus were used as experimental materials.The leaves at the three-leaf stage were taken at 0h（CK）and 7d after alkaline salt stress to prepare semi-thin sections.The leaves of two lines were observed by transmission electron microscopy at 0h（CK）,the mesophyll cells were structurally complete,with many intracellular organelles and normal morphology;after alkaline salt NaHCO₃ stress for 7 days,the chloroplasts of the mesophyll cells of the two lines were destroyed,and the starch granules on the chloroplasts became larger.The transparent and osmium particles increased significantly;the organelles in the cells were reduced or dissolved;in terms of membrane structure,the integrity of the mesophyll cells of the salt-tolerant lines was stronger than that of the salt-sensitive line 1423,and no plasma wall separation was observed.2. Transcriptome analysis of seedlings leaves in B.napus L75m M NaHCO₃ solution was used to treat the salt-tolerant line 2205 and the salt-sensitive line 1423 at the three-leaf stage,and the leaves of each plant were taken for transcriptome sequencing at 0,12,and 24 hours after stress.The results showed that 1802and 6726 differentially expressed genes（DEGs）were identified within and between the two lines,,of which 277 DEGs were common differentially expressed genes within and between lines in response to alkali salt stress;co-expression pattern analysis showed that the salt-sensitive line 1423 has a delayed transcriptional change in response to alkaline salt compared with salt-tolerant line 2205;the GO enrichment of DEGs found that there is a difference in the GO enrichment of the two lines involved in the response to alkaline salt stress,and 58 and 37 GO terms were significantly enriched to the salt-tolerant line 2205and salt-sensitive lines 1423,respectively;KEGG enrichment analysis showed that the salt tolerance pathways enriched in the two lines were different.The salt-tolerant lines were mainly enriched in four pathways:plant hormone signal transduction pathway,starch and sucrose metabolism,endoplasmic reticulum protein process,and amino sugar and nucleotide sugar metabolism pathways;sensitive salt lines were mainly enriched in starch and sugar metabolism and phenylpropane biosynthesis;significant enrichment between the two lines was in carbon metabolism way.3.Distribution of alkaline salt related genes on chromosomesCombined with the information of the salt-tolerant QTL mapping reported in the previous study,277 common DEGs dentified within and between the two lines were mapped to the existing QTL intervals.Thirty-six DEGs were identified on the three chromosomes,A02,A07 and A09,which may be specific to alkaline salt tolerance（NaHCO₃）.Among them,eight and eleven alkaline salt tolerance genes were mapped on A02 and A07 chromosome,respevtively,and the remaining 17 alkaline salt tolerance genes were identified on A09.4.Correlation analysis of physiological indexes of salt tolerance and gene expressionSeedlings at three-leaf stage in B.napus were transferred to Hoagland nutrient solution containing 75mmol/L NaHCO₃ for 0,12,and 24 hours to measure the salt tolerance related indicators.The results showed that the electrical conductivity,antioxidant enzyme（SOD,POD）activity,the contents osmotic adjustment substances（proline,soluble protein and soluble sugar）in the leaves of seedlings gradually increased with the prolongation of the stress time.And the activities of antioxidant enzymes and osmoregulation substances in salt tolerant line 2205 were higher than those in salt sensitive line 1423,while the electrical conductivity was lower than that in salt sensitive line1423.A peroxisomal-related gene（BnaC08g23150D）,three proteins synthesis-related genes（BnaA03g17100D,BnaA02g06190D and BnaA09g00710D）and four starch and sugar metabolism related genes（BnaCnng23260D,BnaA09g00710D,BnaC01g15870D and BnaC03g12060D）with the same change trend in gene expression and physiological indicators were identified,,indicating that the changes of gene expression is closely related to the change of physiological indexes,therefore,it is speculated that the expression of these genes affects the changes of salt tolerance related physiological indicators.5. Cloning and analysis of alkaline salt tolerance geneNine alkaline salt candidate genes were identified through the correlation analysis of physiological indexes and gene expression.And ten highly expressed alkaline salt candidate genes were detected in the QTLs regions;Through GO enrichment of transcriptome and KEGG pathway analysis,40 genes with high expression related to alkaline salt tolerance were obtained.Finally,a total of 59 candidate genes were identified by the above three methods.A salt-tolerant candidate gene BnaA06g13250D was cloned in the two parents 2205 and 1423 using homologous cloning method.The ORF of this gene is765bp,encoding 254 amino acids.Two copies were isolated in 2205,with the isoelectric points of 9.85 and 9.71,and the molecular weight is 27.30k Da and 27.24k Da,respectively;One copy in 1423 was cloned,whit a isoelectric point of 9.78,and the molecular weight is27.34k Da.This gene has two typical domains:the TIFY domain and the CCT_-2 domain.Thirty single nucleotide differences were detected between Bna2205-1 and Bna1423 by DNAMAN software,resulting in 25 amino acid changes and 13 amino acid changes in the conserved domain;two single nucleotides were identified between Bna2205-2 and Bna1423,resulting in one amino acid change,and no amino acid change was identified in the conserved domain.BnaA06g13250D gene was highly expressed in the leaves of the salt-tolerant line 2205,and lower in the salt sensitive line 1423,which indicates that the expression of BnaA06g13250D in 2205 is strongly induced.Analysis of cis-acting elements showed that some cis-acting elements（light-responsive cis-acting regulatory elements,low-temperature response cis-acting elements,elongin response element,cis-acting element involved in abscisic acid reactivity,cis-acting element involved in salicylic acid reactivity,and cis-acting element involved in defense and stress response）are related to plant stress,indicating that BnaC06g31830D may be related to abiotic stress.